Compartmentalization methods based on water-in-oil emulsions have recently been developed for the use in repertoire selection methods (Griffiths98/Ghadessy 01/Sepp02/Griffiths). Compartmentalization segregates individual genes and their encoded products (delivered either as cells (Ghadessy) or expressed in situ (Griffiths/Sepp)) into discrete, physically separate aqueous compartments, thus ensuring the linkage of genotype and phenotype during the selection process. After selection, genes encoding the desired enzymatic activities are isolated either through modification (e.g. methylation), by display on beads (Sepp/Griffiths) or by amplification (Ghadessy 01).
Amplification (CSR) and bead capture (IVC) have been exemplified for the selection of novel enzymatic activities, i.e. variants of Taq polymerase that are either more theiinostable or resistant to the inhibitor heparin (Ghadessy 01) or variants of phosphotriesterase (Griffiths 03) that show increased turn-over. However, both methods depend on high catalytic turn-over (and/or processivity in the case of polymerases) and appear to be poorly suited for the selection of enzymes with a low turnover. While strong selective pressure for a high enzymatic turnover is desirable as an end-point it limits the type of catalytic activities that can be accessed using the system. In particular, starting even from a highly active enzyme, substantial modifications of substrate specificity or even catalytic mechanism are likely to result in much reduced catalytic turn-over, because high-activity enzymes may be many mutations away from the starting sequence and may therefore not be accessible within the limits of the molecular repertoires that can be handled realistically by CSR (or IVC) (1010). For example: From kinetic studies of E. coli DNA polymerase I, mutations such as E710A increased affinity and incorporation of ribonucleotides at the expense of lower catalytic rates and less affinity for wild-type substrates (deoxyribonucleotides) (1). The corresponding mutant of Taq DNA polymerase I, E615A, could incorporate ribonucleotides more efficiently than wild-type polymerase. However, it was only able to synthesize very short fragments and not the full-length Taq gene (J. L. Ong, P. H. unpublished results). In another selection experiment in which Beta-glucuronidase was evolved into a Beta-galactosidase, the desired phenotype was obtained after several rounds of selection but at the expense of catalytic activity. It was also found that selected variants in the initial rounds of selection were able to catalyze the conversion of several different substrates not utilized by either parental enzyme, and at much lower catalytic rates (2). Thus, for many selection objectives (e.g. altered substrate specificity) it is likely that intermediates along the evolutionary trajectory to the new phenotype will have reduced catalytic activity. It would therefore be desirable to have a method for selection of polymerase (and other enzymatic) activities with a lower threshold of selection (ideally requiring just a single turnover event).
For the selection of polymerases with lower catalytic activity or processivity, the present inventors had previously proposed a modification of CSR called short-patch CSR (spCSR) in which only, a small region (a “patch”) of the gene under investigation is randomized and replicated (see original CSR patent). spCSR has allowed the selection of variants of Taq polymerase capable of utilizing ribonucleotide instead of deoxiribonucleotide triphosphates as substrates, which could not be isolated using standard CSR. However, spCSR still requires hundreds to thousands of turnover events for the enzyme to become selectable.
Theoretically, the method of CSR using biotin labelled nucleotides (described in general in PCT/GB98/01889) might be used to detect single turnover events of enzymes, for example polymerases. However in practice the present inventors have found that this method is not optimally efficient for several reasons:                In situ expression of polymerases (inside compartments) from a linear DNA fragment comprising polymerase gene and for example a T7 promotor can be achieved using an in vitro transcription/translation system (ivt) (such as are commercially available). However, we have found that the presence of biotinylated nucleotides (Biotin-dNTP) results in tagging of the 3′ ends of the linear fragment with Biotin regardless of the activity of the expressed polymerase and regardless of the nature of the 3′ end (5′ overhang, blunt, or 3′ overhang). This results in such high-level background that a single-turnover event of a polymerase of interest is not detectable above it.        
The present inventors consider that the reasons for this are:                that some ivt extracts themselves contain endogenous terminal transferase (TT) activity,        T7 RNA pol itself has substantial TT activity (see e.g. McGinness et al (2002) Chem. Biol., 9, 585-596) and is present even before any polymerase of interest has been expressed and so has a head-start in modifying free 3′ ends        DNA polymerases (as far as tested by the present inventors) are poorly expressed by ivt systems,        DNA polymerases are poorly active in the ivt buffers (as far as tested by the present inventors).        
Possible technical solutions to this include the following:    1) conditionally blocked 3′ ends. The problem with this approach is that the chemistry is challenging. In addition, this method does not solve the problem of the polymerases being poorly expressed by ivt systems and being poorly active in ivt buffers.    2) The 2-step method described in PCT/GB01/04108 in the name of the present inventors: 2-step ivt followed by testing for the resultant polymerase to extend DNA. The disadvantage with this method is that it is time-consuming to perform. In addition, it does not solve the problem that DNA polymerases are poorly expressed by ivt systems.
Other methods for the selection of polymerases, include the method of “proximity coupling” used in phage display. Such a method involves the proximal display of both substrate and enzyme on the phage particle (Neri 99, Schultz 00). This concept relies on the in cis conversion of substrate to product or in the case of polymerases the incorporation of a tagged nucleotide into a template-primer duplex substrate tethered to the phage particle (Jestin01, Xia 02). Recently, the method has been used successfully to select for a variant of the Taq polymerase Stoffel fragment that incorporates ribonucleoside triphosphates (rNTPs) with efficiencies approaching those of the wild-type enzyme for dNTP substrates (Xia 02). However, there are several problems associated with the use of this method. Importantly, selection conditions have to be compatible with phage viability and the intramolecular tethering of the substrate may favor the selection of polymerases with low affinity for template-primer duplex and poor processivity.
Therefore, there remains a need in the art for the provision of a method for the selection of nucleic acid processing molecules, in particular DNA polymerases which possess a low catalytic turnover and/or processivity, which method is not constrained by selection conditions which are required for phage viability.